The present invention, in some embodiments thereof, relates to polymer-protein conjugates and, more particularly, but not exclusively, to polymer-protein conjugates which form a scaffold, to processes of generating same and to uses thereof in, for example, tissue engineering.
As the field of tissue engineering evolves, there is a need for new biomaterial scaffolds that can provide more than just architectural and mechanical support. New “hybrid” materials are being developed as sophisticated scaffolds wherein biological polymers such as alginate, collagen or fibrinogen are combined with synthetic polymers to provide added versatility and bioactivity at the material/cell interface. From the perspective of cellular interactions, the biological domains of the hybrid material may actively participate in stimulating cells towards the formation of functional tissues. Bioactive signals are controlled via biological macromolecules such as protein segments [Cutler and Garcia, Biomaterials 2003, 24:1759-1770], growth factors [Seliktar et al., J Biomed Mater Res A 2004, 68:704-716; Zisch et al., FASEB J 2003; 17:2260-2262; DeLong et al., Biomaterials 2005, 26:3227-3234] or short bioactive peptides [Mann et al., Biomaterials 2001, 22:3045-3051; Lutolf et al., Proc Natl Acad Sci USA 2003, 100:5413-5418; Stile and Healy, Biomacromolecules 2001, 2:185494]. These elements are capable of influencing cell migration, proliferation, and guided differentiation [Dikovsky et al., Biomaterials 2006, 27:1496-1506]. From the perspective of biomaterial properties, “smart” polymers may also be used to provide better control over bulk features of the scaffold in response to changes in temperature, pH, or light [Furth et al., Biomaterials 2007, 28:5068-5073; Galaev and Mattiasson, Trends Biotechnol 1999, 17:335-340]. Hybrid materials made with smart polymers have additional degrees of freedom, including control over bulk density, degradability, porosity and compliance, all of which can be regulated by the synthetic polymer component [Peppas et al., Annu Rev Biomed Eng 2000, 2:9-29; Tsang and Bhatia, Adv Drug Deliv Rev 2004, 56:1635-1647; 3] Baier Leach et al., Biotechnol Bioeng 2003, 82:578-589].
Hybrid materials have been prepared based on conjugation of endogenous proteins with versatile synthetic polymers [Almany and Seliktar, Biomaterials 2005, 26:2467-2477; Gonen-Wadmany et al., Biomaterials 2007, 28:3876-3886; Peled et al., Biomed Mater Res A 2007, 80:874-884; Seliktar, Ann NY Acad Sci 2005, 1047:386-394]. The effect of alternating structural properties of hydrogels made from poly(ethylene glycol) (PEG) conjugated to fibrinogen on the morphology and remodeling of encapsulated smooth muscle cells has been investigated [Dikovsky et al., Biomaterials 2006, 27:1496-1506; Dikovsky et al., Biophys J 2008, 94:2914-2925]. These materials exhibited an ability to control cellular behavior by changing factors such as density, stiffness, and proteolytic degradability through the versatile synthetic component. The fibrinogen is a natural substrate for tissue remodeling which contains several cell signaling domains, including a protease degradation substrate and cell adhesion motifs [Herrick et al., Int J Biochem Cell Biol 1999, 31:741-746; Werb, Cell 1997, 91:439-442].
International Patent Application PCT/IL2004/001136 (published as WO2005/061018) and U.S. patent application Ser. No. 11/472,437 describe a biodegradable scaffold composed of a protein (e.g., fibrinogen) backbone cross-linked by a synthetic polymer such as poly(ethylene glycol), and methods of generating such scaffolds from polymer-protein conjugates.
International Patent Application PCT/IL2008/000521 (published as WO 2008/126092) describes scaffolds composed of albumin or thiolated collagen cross-linked by a synthetic polymer such as poly(ethylene glycol).
Reverse thermo-responsive polymers are capable of producing low viscosity aqueous solutions at ambient temperature, and forming a gel at a higher temperature. This property may be used to generate implants in situ [Cohn et al., Biomacromolecules 2005, 6:1168-1175].
Stile and Healy [Biomacromolecules 2001, 2:185-194] modified a smart polymer, N-isopropylacrylamide, with RGD (Arg-Gly-Asp) containing peptides to form a reversible thermo-sensitive hydrogel with bioactive segments for cell culture studies. They reported that the conjugation of RGD peptides to the thermo-responsive smart polymer does not compromise the temperature-induced sol-gel transition of the hydrogels. They further reported that the conjugated RGD peptide enhanced the biological interactions of the otherwise inert N-isopropylacrylamide polymer network.
Reverse thermo-responsive polymers having a poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO tri-block structure, referred to as “poloxamers”, have also been reported. The endothermic sol-gel transition takes place due to an increase in entropy caused by release of water molecules bound to the PPO segments as temperature increases [Alexandridis, Colloid Surface A 1995, 96:1-46].
Pluronic® F127 poloxamer is a well known synthetic triblock copolymer (PEO99-PPO67-PEO99) [Nagarajan and Ganesh, J Colloid Interface Sci 1996, 184:489-499; Sharma and Bhatia, Int J Pharm 2004, 278:361-377; Cohn et al., Biomaterials 2003, 24:3707-3714], that exhibits a reverse thermal gelation (RTG) property above a critical temperature in aqueous solutions. Cohn et al. [Polym Adv Tech 2007; 18:731-736] reported that polymerized F127 displays reverse thermal gelation at lower concentrations and with enhanced mechanical properties, as compared with F127.
Additional background art includes Halstenberg et al. [Biomacromolecules 2002, 3:710-723], Cohn et al. [Polym Adv Tech 2007; 18:731-736], and U.S. Pat. No. 7,842,667.